Recent advances in imaging and single molecule fluorescence microscopy (SMFM) have enabled direct observation of biological processes at the molecular level.1-3 Fluorescence-based imaging techniques rely on bright and photostable fluorescent probes with robust photophysical properties. Currently available fluorescent probes for biological labeling include fluorescent proteins,2,4 organic dyes1,5 and quantum dots (QDs).6 Single organic dye molecules can be small in size (<1 nm), which facilitates non-perturbative biomolecule labeling. In addition, the photophysical properties of organic dyes can be enhanced using reducing agents and oxygen scavenger systems.7-9 Recently, single organic dyes have enabled super-resolution imaging techniques such as stochastic optical reconstruction microscopy (STORM).10 QDs are exceptionally bright, multi-color fluorescent probes, however, QD fluorescence emission generally exhibits transient “blinking” behavior.11 Compared to single dye molecules, QDs are relatively large (˜10-20 nm) due to their core/shell structure and further surface modification for biocompatability, which may complicate in vivo labeling and biomolecule conjugation.
Accordingly, there is a strong need to develop bright and photostable nanoscale probes for biological imaging. There is also a need for new fluorescence-based imaging compositions and techniques that provide more robust fluorescent probes than currently used fluorescent probes. Compositions and techniques that provide improved photophysical properties of fluorescent tags are also needed to advance the field of imaging and biomolecular analysis.